N-acetylglucosamine, often identified as GlcNAc, is a monosaccharide derivative of glucose that modifies proteins of eukaryotic organisms. One form of modification, referred to as O-GlcNAc (O-linked β-N-acetylglucosamine) glycosylation, occurs by the addition of a single N-acetylglucosamine residue to serine/threonine residues. This modification was long thought to exist solely on nuclear and cytosolic proteins, but it has been recently discovered on extracellular proteins. Unlike other types of glycosylation, the sugar residue is not elongated into complex oligosaccharides. In fact, O-GlcNAc glycosylation shares many features with protein phosphorylation, a fundamental mechanism for intracellular communication, and it has been postulated that O-GlcNAc glycosylation has a mutually exclusive relationship with phosphorylation. O-GlcNAc glycosylation is involved in many cellular processes such as nutrient sensing, stress response, transcription, translation, cell signaling and cell cycle regulation. O-GlcNAc glycosylation is also involved in many human diseases including diabetes, Alzheimer's disease and cancer.
An alternative form of GlcNAc glycosylation, N-GlcNAc (N-linked β-N-acetylglucosamine) glycosylation, is also known. N-GlcNAc modification of proteins occurs on arginine residues and has been observed in bacteria, for example.
Despite the importance of GlcNAc glycosylation, detection of GlcNAc glycosylation is a challenging task. O-GlcNAc glycosylation has no effect on protein mobility during electrophoresis because of its small size and neutral charge. Traditionally, antibodies and lectins have been applied to detect O-GlcNAc, but these reagents suffer from weak binding affinity and limited specificity for O-GlcNAc. Recently, methods for labeling O-GlcNAc with radioisotope tagged or chemically modified galactose using recombinant galactosyltransferases have been reported. For example, 3H tagged galactose, Keto-Gal, or GalNAz were covalently linked to O-GlcNAc using galactosyltransferases or mutant galactosyltransferase Gal-T1 (Y289L). However, detection of O-GlcNAc glycosylation remains difficult. Chemoenzymatic labeling with mutant galactosyltransferase Gal-T1(Y289L) has also been applied to detect N-GlcNAc. In addition, mass spectrometry has been applied to detect GlcNAc modification in general.